S. saprophyticus is a major cause of community-acquired UTI in young women. Knowledge of the virulence mechanisms of S. saprophyticus has advanced in recent years, particularly with the acquisition and analysis of whole genome sequence data. The majority of acknowledged virulence factors of S. saprophyticus are proteins tethered to the cell surface, which with the exception of the Ssp lipase , are all involved in adhesion: Aas is an autolysin that also binds to fibronectin ; UafA adheres to uroepithelial cells via an unidentified ligand ; SdrI binds to collagen I and fibronectin [9, 31] and UafB binds to fibronectin, fibrinogen and urothelial cells . Here we have identified another cell wall-anchored protein produced by S. saprophyticus that we have termed SssF - the sixth surface protein described for this species.
The sssF gene was identified in the sequence of the pSSAP2 plasmid of S. saprophyticus MS1146 due to the presence of the canonical LPXTG sortase motif in the translated protein sequence. A copy of the sssF gene is also located on the pSSP1 plasmid of S. saprophyticus ATCC 15305 (99% nucleotide identity; Figure 1), but it was not acknowledged as encoding an LPXTG motif-containing protein . We recently characterised another plasmid-coded LPXTG motif-containing protein of S. saprophyticus MS1146, UafB, as an adhesin . We first sought to investigate whether SssF was another adhesin, since a considerable proportion of characterised Gram-positive covalently surface anchored proteins have adhesive functions , including every other known S. saprophyticus LPXTG motif-containing protein. No evidence of an adhesion phenotype for SssF was detected.
SssF protein sequence searches with the BLAST database provided an output of uncharacterised staphylococcal proteins with a maximum 39% amino acid identity to SssF across the entire protein sequence, mostly annotated as hypothetical cell wall-anchored proteins. In contrast to S. saprophyticus, the genes encoding these SssF-like proteins are located on the chromosome, rather than on a plasmid, in every other sequenced staphylococcal species. Some of these staphylococcal SssF-like proteins contain atypical sortase motifs. At this stage it is not known whether all of these proteins are sorted to the cell surface efficiently, but SasF has been shown to be associated with the cell wall of S. aureus 8325-4 even with the non-classical LPKAG sortase motif . There was a distinct lack of phenotypic data for these SssF-like proteins until a role for SasF was recently uncovered. Kenny et al.  observed that sasF was the most upregulated gene in S. aureus MRSA252 microarray and qRT-PCR experiments upon challenge with linoleic acid. The protective function of SasF was apparent when examined in a linoleic acid emulsion agar plate-based bacterial survival assay. Our hypothesis focused on the possibility that SssF possessed a similar function to SasF, but no linoleic acid resistance phenotype for SssF was observed in the S. saprophyticus MS1146 genetic background. Using the linoleic acid emulsion agar plate bacterial survival assay in the presence 0.85 M NaCl, we observed a higher survival amongst S. saprophyticus strains that harbour the sssF gene than those that lack sssF. We then successfully expressed SssF heterologously in a S. aureus SH1000sasF host and demonstrated restored resistance to linoleic acid. We found S. saprophyticus MS1146 to be intrinsically more resistant to linoleic acid than S. aureus SH1000. This remains to be explored but could be due to a number of species/strain specific factors including the action of redundant S. saprophyticus MS1146 resistance mechanisms or variations in surface components such as capsule or teichoic acids.
We found that the survival of S. aureus SH1000 and its derivatives was markedly increased in the presence of linoleic acid at pH 6.0 compared to pH 7.4. This result is consistent with previous studies of the staphylococcal fatty acid modifying enzyme (FAME), an unidentified but partially characterised protein secreted by most staphylococci which detoxifies free fatty acids by esterifying them to an alcohol [34, 35]. The FAME of S. aureus and S. epidermidis demonstrate optimal activity at pH 6.0, and have little activity at pH 7.4 [35, 36]. This is congruent with human skin having a slightly acidic pH of 5.5-6 . RP-HPLC experiments using linoleic acid and crude protein extracts demonstrated that SssF activity is distinct from FAME activity (data not shown). Other antimicrobial fatty acids such as sapienic acid have yet to be examined as substrates for SssF or SasF. We hypothesise that some or all of the other uncharacterised SssF-like proteins exhibit fatty acid resistance activity, but this remains to be demonstrated experimentally.
There are precedents for bacterial surface structures that provide protection against bactericidal free fatty acids. Gram-positive bacterial cell wall teichoic acids provide protection against free fatty acid mediated killing of S. aureus . The IsdA protein of S. aureus reduces bacterial hydrophobicity when expressed at the cell surface under the cue of iron starvation to resist fatty acid membrane attack and also promotes fatty acid resistance of S. aureus in a volunteer human skin survival model . Our studies however found that expression of SssF does not influence cell surface hydrophobicity of S. saprophyticus, and this corresponds with matching data for SasF and S. aureus .
No conserved motifs that might predict the functional residues of SssF-like proteins were identified. The observation that the SssF-like proteins are structurally related to myosin is noteworthy, especially in light of the recent characterisation of myosin cross-reactive antigens of Streptococcus pyogenes and Bifidobacterium breve as fatty acid hydratases [40, 41]. These enzymes act to detoxify unsaturated free fatty acids, including linoleic acid. Homologous proteins with modest primary sequence identity but similar tertiary structures are acknowledged in both bacterial  and mammalian  lipid-binding protein families. It is possible that conserved tertiary protein structure between SssF-like proteins contributes to their function.
S. saprophyticus is a uropathogen, but SssF is unlikely to have evolved to facilitate survival in the urinary tract. A common trait of staphylococci is skin colonisation. Staphylocidal free fatty acids (especially unsaturated) are present on human skin  and are also active in staphylococcal abscesses . Furthermore, linoleic acid is one of the most abundant polyunsaturated fatty acids on human skin , and is also present in vaginal secretions . SssF may be an important determinant for survival of S. saprophyticus in the events preceding urethral entry in community-acquired UTI - colonisation of perineal and periurethral tissue. This would account for the absence of SssF involvement in the mouse model of UTI, in which the inocula are delivered directly into the bladder.
The location of sssF on a plasmid in both sequenced S. saprophyticus strains is intriguing, particularly as every other staphylococcal SssF-like protein is chromosomally encoded. It has been observed that many genes that are located on plasmids encode for traits which have extracellular functions , and sssF falls into this category. Furthermore, plasmid genes have often been noted to confer selective advantage to the bacteria in some environmental niches but not others . Every pathogenic staphylococcal species known to carry a chromosomal sssF-like gene is known to commensally inhabit the skin, and this can be considered their main niche. S. saprophyticus, on the other hand, primarily resides in the genitourinary and gastrointestinal tracts [4, 20]. It is feasible that since human skin is not the major habitat of S. saprophyticus, sssF has been retained as an accessory gene required for survival on the skin during non-UTI periods. Nonetheless, it may still be the case that sssF is found on the chromosome of some S. saprophyticus strains.
SssF represents the fourth LPXTG motif-containing protein described in S. saprophyticus. We present here evidence that the S. saprophyticus SssF protein has a role in the protection against free fatty acid mediated killing, and that it is a member of a newly identified protein family broadly distributed throughout the Staphylococcus genus.